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Corneal blindness affects more than 5 million individuals, with over 180,000 corneal transplantations (CTs) performed annually. In high-risk CTs, almost all grafts are rejected within 10 years. Here, we investigated adeno-associated virus (AAV) ex vivo gene therapy to establish immune tolerance in the corneal allograft to prevent high-risk CT rejection. Our previous work has demonstrated that HLA-G contributes to ocular immune privilege by inhibiting both immune cells and neovascularization; however, homodimerization is a rate-limiting step for optimal HLA-G function. Therefore, a chimeric protein called single-chain immunomodulator (scIM), was engineered to mimic the native activity of the secreted HLA-G dimer complex and eliminate the need for homodimerization. In a murine corneal burn model, AAV8-scIM significantly reduced corneal vascularization and fibrosis. Next, ex vivo AAV8-scIM gene delivery to corneal allografts was evaluated in a high-risk CT rejection rabbit model. All scIM-treated corneas were well tolerated and transparent after 42 days, while 83% of vehicle-treated corneas were rejected. Histologically, AAV-scIM-treated corneas were devoid of immune cell infiltration and vascularization, with minimal fibrosis at the host-graft interface. The data collectively demonstrate that scIM gene therapy prevents corneal neovascularization, reduces trauma-induced corneal fibrosis, and prevents allogeneic CT rejection in a high-risk large animal model., Competing Interests: Declaration of interests M.L.H. and R.B.S. are co-inventors of the scIM technology evaluated here and are listed on a patent (pending) owned by Texas Tech University and UNC-Chapel Hill. M.L.H., R.B.S., C.L., and B.C.G. are co-founders of Bedrock Therapeutics, which has licensed the scIM technology evaluated here., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Ocular graft versus host disease (OGvHD) develops after allogeneic hematopoietic stem cell transplantation (HSCT) and manifests as ocular surface inflammatory disease. This study evaluated the efficacy of adeno-associated virus (AAV) gene therapy encoding human leukocyte antigen G (HLA-G) to inhibit OGvHD. A major histocompatibility mismatch chronic OGvHD murine model was evaluated. 7 days after HSCT, mice were dosed subconjunctivally with scAAV8-HLA-G1/5 (1 x 10 9 vg/eye), topical cyclosporine (twice daily), or left untreated. Body weights and tear production (red thread test) were recorded, and eyelid, corneal opacity, and corneal fluorescein retention were scored through day 44 after HSCT. Tissues were collected for vector biodistribution, ocular histology, and immunofluorescence. Compared with untreated HSCT eyes, those dosed with scAAV8-HLA-G1/5 had significantly reduced clinical inflammatory signs of OGvHD. On histology, eyes that received scAAV8-HLA-G1/5 or cyclosporine had a significantly lower mean limbal mononuclear cell count when compared with non-treated HSCT eyes. HLA-G immunofluorescence was detected in the subconjunctiva and peripheral cornea in HSCT animals treated with scAAV8-HLA-G1/5. Vector genomes were detected in the lacrimal gland, but not in the other tested organs. These results provide evidence that subconjunctival AAV targets ocular surface and corneal disease and support that HLA-G-based gene therapy may be an effective treatment for OGvHD., Competing Interests: M.L.H. and B.C.G. are coinventors of the AAV-HLA-G technology evaluated herein and are listed on a patent (pending) that is owned by the University of North Carolina and North Carolina State University. M.L.H. and B.C.G. are co-founders of Astro Therapeutics (which has licensed the HLA-G technology evaluated herein) and co-founders of Bedrock Therapeutics., (© 2023 The Author(s).)

Equine recurrent uveitis (ERU) is a spontaneous, painful, and vision threatening disease affecting up to 25% of equine populations worldwide. Current treatments of ERU are non-specific and have many side effects which limits them to short-term use. In order to develop an effective therapy for ERU, we investigated the use of adeno-associated virus (AAV) gene therapy, exploiting a natural immune tolerance mechanism induced by equine interleukin-10 (Equine-IL10). The purpose of this study was to evaluate the therapeutic efficacy of a single intravitreal (IVT) dose of AAV8-Equine-IL10 gene therapy for inhibition of experimental autoimmune uveitis (EAU) in rats. Each rat was dosed intravitreally (IVT) in both eyes with either balanced salt solution (BSS) (control; n = 4), AAV8-Equine-IL10 at a low dose (2.4x109 vg; n = 5) or high dose (2.4x1010 vg; n = 5). EAU was induced in all groups of rats 7 days after IVT injections and euthanized 21 days post-injection. Ophthalmic examination and aqueous humor (AH) cell counts were recorded with the observer blinded to the treatment groups. Histopathology and qPCR were performed on selected ocular tissues. Data presented herein demonstrate that AAV8-Equine-IL10 treated rats exhibited a significant decrease in clinical inflammatory scores and AH cell counts compared to BSS-treated EAU eyes on days 10, 12 and 14 post EAU induction at both administered vector doses. Mean cellular histologic infiltrative scores were also significantly less in AAV8-Equine-IL10 dosed rats compared to the BSS group. Intravitreal injection of AAV8-Equine-IL10 resulted in Equine-IL10 cDNA expression in the ciliary body, retina, cornea, and optic nerve in a dose-dependent manner. A single IVT injection of AAV8-Equine-IL10 appeared to be well-tolerated and inhibited EAU even at the lowest administered dose. These results demonstrate safety and efficacy of AAV8-Equine-IL10 to prevent EAU and support continued exploration of AAV gene therapy for the treatment of equine and perhaps human recurrent uveitis., Competing Interests: The authors have declared that no competing interests exist.

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ease of accessibility via multiple administration routes. Subconjunctival (SC) injections offer advantages over other intraocular administration routes as they are simple, safe, require only local anesthesia, and are usually performed in an outpatient setting. Although SC injections in small animals usually require the assistance of an operating microscope due to the size of the eye, they are widely utilized for drug delivery, including gene therapy vectors. Previous work has demonstrated that SC injection of specific adeno-associated virus (AAV) serotypes is a valid gene delivery strategy for targeted transduction of the ocular surface, eye muscle, cornea, and optic nerve, providing a potential approach for the treatment of many ocular diseases. Herein, a detailed protocol is presented for SC injections in a mouse model using an injection system consisting of a programmable infusion/withdrawal syringe pump (which allows for consistent and precise injection speed and pressure) and a gastight removable syringe coupled with microinjection needles. The injection system is also adaptable for other intraocular administration routes such as intrastromal, intracameral, intravitreal, and subretinal injections in small animals. Although the delivery of adeno-associated viral vectors for ocular gene therapy studies is described, the protocol herein can also be adapted for a variety of ophthalmic solutions in small animal models. The key practical steps in the administration route, setup for the injection platform, preparation of the injection, and tips from direct experience will be discussed in detail. In addition, common validation techniques for AAV delivery confirmation to the desired tissues will also be briefly discussed.

Adeno-associated virus (AAV) was first characterized as small "defective" contaminant particles in a simian adenovirus preparation in 1965. Since then, a recombinant platform of AAV (rAAV) has become one of the leading candidates for gene therapy applications resulting in two FDA-approved treatments for rare monogenic diseases and many more currently in various phases of the pharmaceutical development pipeline. Herein, we summarize rAAV approaches for the treatment of diverse types of cancers and highlight the natural anti-oncogenic effects of wild-type AAV (wtAAV), including interactions with the cellular host machinery, that are of relevance to enhance current treatment strategies for cancer.

The chronic ocular toxicity, tolerability, and inflammation following corneal intrastromal injection of saline or escalating doses of an adeno-associated virus (AAV) containing a codon-optimized α-l-iduronidase (AAV-opt- IDUA ) expression cassette were evaluated in New Zealand White rabbits. Corneal opacity following corneal intrastromal injection resolved by 24 h. Mild elevation of clinical ocular inflammation was observed 24 h after injection, but it returned to baseline by day 7 and no abnormalities were noted through 6 months of observation after injection. Vector genomes and IDUA cDNA were detected in the injected corneas in a dose-dependent manner. Both the lowest administered AAV-opt- IDUA dose, shown to be effective in mucopolysaccharidosis type I (MPS I) dogs, and a 10-fold higher dose of AAV-opt- IDUA resulted in no detectable immunologic response or adverse effect in rabbits. Vector genomes outside of the eye were rarely detected following corneal intrastromal injection of AAV-opt- IDUA , and neutralizing antibodies to the AAV capsid were not present at the experimental conclusion. This study, combined with our previous studies in MPS I dogs, suggests that AAV-opt- IDUA corneal gene therapy following corneal intrastromal injection of AAV-opt- IDUA has the potential to prevent and reverse blindness in MPS I patients in a safe and effective manner., (© 2020 The Author(s).)

Gene delivery approaches using adeno-associated virus (AAV) vectors are currently the preferred method for human gene therapy applications and have demonstrated success in clinical trials for a diverse set of diseases including retinal blindness. To date, no clinical trials using AAV gene therapy in the anterior eye have been initiated; however, corneal gene delivery appears to be an attractive approach for treating both corneal and ocular surface diseases. Multiple preclinical studies by our lab and others have demonstrated efficient AAV vector-mediated gene delivery to the cornea for immunomodulation, anti-vascularization, and enzyme supplementation. Interestingly, the route of AAV vector administration and nuances such as administered volume influence vector tropism and transduction efficiency. In this chapter, a detailed protocol for AAV vector production and specific approaches for AAV-mediated gene transfer to the cornea via subconjunctival and intrastromal injections are described.

Non-infectious uveitis (NIU) is an intractable, recurrent, and painful disease that is a common cause of vision loss. Available treatments of NIU, such as the use of topical corticosteroids, are non-specific and have serious side effects which limits them to short-term use; however, NIU requires long-term treatment to prevent vision loss. Therefore, a single dose therapeutic that mediates long-term immunosuppression with minimal side effects is desirable. In order to develop an effective long-term therapy for NIU, an adeno-associated virus (AAV) gene therapy approach was used to exploit a natural immune tolerance mechanism induced by the human leukocyte antigen G (HLA-G). To mimic the prevention of NIU, naïve Lewis rats received a single intravitreal injection of AAV particles harboring codon-optimized cDNAs encoding HLA-G1 and HLA-G5 isoforms one week prior to the induction of experimental autoimmune uveitis (EAU). AAV-mediated expression of the HLA-G-1 and -5 transgenes in the targeted ocular tissues following a single intravitreal injection of AAV-HLA-G1/5 significantly decreased clinical and histopathological inflammation scores compared to untreated EAU eyes (p < 0.04). Thus, localized ocular gene delivery of AAV-HLA-G1/5 may reduce the off-target risks and establish a long-term immunosuppressive effect that would serve as an effective and novel therapeutic strategy for NIU, with the potential for applications to additional ocular immune-mediated diseases.

Genomic instability is a hallmark of breast cancer, contributes to tumor heterogeneity, and influences chemotherapy resistance. Although Gap 2 and mitotic checkpoints are thought to prevent genomic instability, the role of these checkpoints in breast cancer is poorly understood. Here, we assess the Gap 2 and mitotic checkpoint functions of 24 breast cancer and immortalized mammary epithelial cell lines representing four of the six intrinsic molecular subtypes of breast cancer. We found that patterns of cell cycle checkpoint deregulation were associated with the intrinsic molecular subtype of breast cancer cell lines. Specifically, the luminal B and basal-like cell lines harbored two molecularly distinct Gap 2/mitosis checkpoint defects (impairment of the decatenation Gap 2 checkpoint and the spindle assembly checkpoint, respectively). All subtypes of breast cancer cell lines examined displayed aberrant DNA synthesis/Gap 2/mitosis progression and the basal-like and claudin-low cell lines exhibited increased percentages of chromatid cohesion defects. Furthermore, a decatenation Gap 2 checkpoint gene expression signature identified in the cell line panel correlated with clinical outcomes in breast cancer patients, suggesting that breast tumors may also harbor defects in decatenation Gap 2 checkpoint function. Taken together, these data imply that pharmacological targeting of signaling pathways driving these phenotypes may lead to the development of novel personalized treatment strategies for the latter two subtypes which currently lack targeted therapeutic options because of their triple negative breast cancer status.

Human embryonic stem (hES) cells activate a rapid apoptotic response after DNA damage but the underlying mechanisms are unknown. A critical mediator of apoptosis is Bax, which is reported to become active and translocate to the mitochondria only after apoptotic stimuli. Here we show that undifferentiated hES cells constitutively maintain Bax in its active conformation. Surprisingly, active Bax was maintained at the Golgi rather than at the mitochondria, thus allowing hES cells to effectively minimize the risks associated with having preactivated Bax. After DNA damage, active Bax rapidly translocated to the mitochondria by a p53-dependent mechanism. Interestingly, upon differentiation, Bax was no longer active, and cells were not acutely sensitive to DNA damage. Thus, maintenance of Bax in its active form is a unique mechanism that can prime hES cells for rapid death, likely to prevent the propagation of mutations during the early critical stages of embryonic development., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The pathogenesis of eosinophilic esophagitis (EoE) is incompletely understood. In certain eosinophilic diseases, activation of tyrosine kinase after fusion of the Fip1-like-1 and platelet-derived growth factor receptor-α genes (F-P fusion gene) mediates eosinophilia via downstream effectors such as extracellular-regulated kinase (ERK1/2) and signal transducers and activators of transcription (STAT5). This mechanism has not been examined in EoE. Our aim was to detect the F-P fusion gene, pERK1/2, and pSTAT5 in esophageal tissue from patients with EoE, gastroesophageal reflux disease (GERD), and normal controls. We performed a cross-sectional pilot study comparing patients with steroid-responsive and steroid-refractory EoE, to GERD patients and normal controls. EoE cases were defined by consensus guidelines. Fluorescence in situ hybridization (FISH) was performed to detect the F-P fusion gene and immunohistochemistry (IHC) was performed to detect pERK1/2 and pSTAT5 in esophageal biopsies. Twenty-nine subjects (median age 30 years [range 1-59]; 16 males; 24 Caucasians) were included: eight normal, six GERD, and 15 EoE (five steroid-refractory). On FISH, 98%, 99%, and 99% of the nuclei in the normal, GERD, and EoE groups, respectively, were normal (P= 0.42). On IHC, a median of 250, 277, and 479 nuclei/mm(2) stained for pERK 1/2 in the normal, GERD, and EoE groups, respectively (P= 0.07); the refractory EoE patients had the highest degree pERK 1/2 staining (846 nuclei/mm(2); P= 0.07). No trend was seen for pSTAT5. In conclusion, the F-P fusion gene was not detected with increased frequency in EoE. Patients with EoE had a trend toward higher levels of pERK 1/2, but not STAT5, in the esophageal epithelium, with highest levels in steroid-refractory EoE patients., (© 2011 Copyright the Authors. Journal compilation © 2011, Wiley Periodicals, Inc. and the International Society for Diseases of the Esophagus.)

Human embryonic stem cells (hESCs) are primed for rapid apoptosis following mild forms of genotoxic stress. A natural form of such cellular stress occurs in response to recombinant adeno-associated virus (rAAV) single-strand DNA genomes, which exploit the host DNA damage response for replication and genome persistence. Herein, we discovered a unique DNA damage response induced by rAAV transduction specific to pluripotent hESCs. Within hours following rAAV transduction, host DNA damage signaling was elicited as measured by increased gamma-H2AX, ser15-p53 phosphorylation, and subsequent p53-dependent transcriptional activation. Nucleotide incorporation assays demonstrated that rAAV transduced cells accumulated in early S-phase followed by the induction of apoptosis. This lethal signaling sequalae required p53 in a manner independent of transcriptional induction of Puma, Bax and Bcl-2 and was not evident in cells differentiated towards a neural lineage. Consistent with a lethal DNA damage response induced upon rAAV transduction of hESCs, empty AAV protein capsids demonstrated no toxicity. In contrast, DNA microinjections demonstrated that the minimal AAV origin of replication and, in particular, a 40 nucleotide G-rich tetrad repeat sequence, was sufficient for hESC apoptosis. Our data support a model in which rAAV transduction of hESCs induces a p53-dependent lethal response that is elicited by a telomeric sequence within the AAV origin of replication.

Topoisomerase IIalpha (topoIIalpha) is an essential mammalian enzyme that topologically modifies DNA and is required for chromosome segregation during mitosis. Previous research suggests that inhibition of topoII decatenatory activity triggers a G(2) checkpoint response, which delays mitotic entry because of insufficient decatenation of daughter chromatids. Here we examine the effects of both topoIIalpha and topoIIbeta on decatenatory activity in cell extracts, DNA damage and decatenation G(2) checkpoint function, and the frequencies of p16(INK4A) allele loss and gain. In diploid human fibroblast lines, depletion of topoIIalpha by small-interfering RNA was associated with severely reduced decatenatory activity, delayed progression from G(2) into mitosis and insensitivity to G(2) arrest induced by the topoII catalytic inhibitor ICRF-193. Furthermore, interphase nuclei of topoIIalpha-depleted cells showed increased frequencies of losses and gains of the tumor suppressor genetic locus p16(INK4A). This study shows that the topoIIalpha protein is required for decatenation G(2) checkpoint function, and inactivation of decatenation and the decatenation G(2) checkpoint leads to abnormal chromosome segregation and genomic instability.

The decatenation G2 checkpoint is proposed to delay cellular progression from G2 into mitosis when intertwined daughter chromatids are insufficiently decatenated. Previous studies indicated that the ATM- and Rad3-related (ATR) checkpoint kinase, but not the ataxia telangiectasia-mutated (ATM) kinase, was required for decatenation G2 checkpoint function. Here, we show that the method used to quantify decatenation G2 checkpoint function can influence the identification of genetic requirements for the checkpoint. Normal human diploid fibroblast (NHDF) lines responded to the topoisomerase II (topo II) catalytic inhibitor ICRF-193 with a stringent G2 arrest and a reduction in the mitotic index. While siRNA-mediated depletion of ATR and CHEK1 increased the mitotic index in ICRF-193 treated NHDF lines, depletion of these proteins did not affect the mitotic entry rate, indicating that the decatenation G2 checkpoint was functional. These results suggest that ATR and CHEK1 are not required for the decatenation G2 checkpoint, but may influence mitotic exit after inhibition of topo II. A re-evaluation of ataxia telangiectasia (AT) cell lines using the mitotic entry assay indicated that ATM was required for the decatenation G2 checkpoint. Three NHDF cell lines responded to ICRF-193 with a mean 98% inhibition of the mitotic entry rate. Examination of the mitotic entry rates in AT fibroblasts upon treatment with ICRF-193 revealed a significantly attenuated decatenation G2 checkpoint response, with a mean 59% inhibition of the mitotic entry rate. In addition, a normal lymphoblastoid line exhibited a 95% inhibition of the mitotic entry rate after incubation with ICRF-193, whereas two AT lymphoblastoid lines displayed only 36% and 20% inhibition of the mitotic entry rate. Stable depletion of ATM in normal human fibroblasts with short hairpin RNA also attenuated decatenation G2 checkpoint function by an average of 40%. Western immunoblot analysis demonstrated that treatment with ICRF-193 induced ATM autophosphorylation and ATM-dependent phosphorylation of Ser15-p53 and Thr68 in Chk2, but no appreciable phosphorylation of Ser139-H2AX or Ser345-Chk1. The results suggest that inhibition of topo II induces ATM to phosphorylate selected targets that contribute to a G2 arrest independently of DNA damage.

Arsenic is a ubiquitous environmental contaminant associated with increased risks of human cancers of the skin, lung, bladder, and prostate. Intriguingly, it is also used to treat certain types of leukemia. It has recently been suggested that these paradoxic effects may be mediated by arsenic's ability to simultaneously activate DNA damage and apoptotic and transformation pathways. Here, we investigate the effects of arsenic exposure on the induction of the growth arrest and DNA damage protein 45 alpha (GADD45 alpha), which is thought to play roles in apoptosis, DNA damage response, and cell cycle arrest. We found that arsenic transcriptionally activates the gadd45 alpha promoter located in a 153-bp region between -234 and -81, relative to the transcriptional start site. In addition, this transcriptional induction was abrogated in the presence of H2O2 scavengers, suggesting a role for H2O2 in the transcriptional control of the gadd45a gene through a Fenton-like free radical mechanism.

The human genome sequence provides researchers with a genetic framework to eventually understand the relationships of gene-environment interactions. This wealth of information has led to the birth of several related areas of research, including proteomics, functional genomics, pharmacogenomics, and toxicogenomics. Developing techniques such as DNA/protein microarrays, small-interfering RNA (siRNA) applications, two-dimensional gel electrophoresis, and mass spectrometry in conjunction with advanced analysis software and the availability of Internet databases offers a powerful set of tools to investigate an individual's response to specific stimuli. This review summarizes these emerging scientific fields and techniques focusing specifically on their applications to the complexities of gene-environment interactions and their potential role in environ-mental biosecurity.

A wide variety of metals have been reported to act as mutagenic and carcinogenic agents in both human and animal studies. The underlying mechanisms are being extensively investigated. Recently, a new sub-discipline of molecular carcinogenesis has surfaced and new techniques and instruments are being developed which allow exploration of the complex biological relationships and signaling pathways involved in response to metal exposure at the molecular level. The 2nd Conference on Molecular Mechanisms of Metal Toxicity and Carcinogenesis was held at NIOSH in Morgantown, West Virginia, Sept. 8-11, 2002. One hundred thirty scientist from sixteen countries presented their novel findings and investigations of metal-induced carcinogenesis. The conference focused on state-of-the-art research and developments in metal toxicity and carcinogenesis. Emphasis was placed on delineating molecular mechanisms involved in free radical effects, cellular uptake, signaling pathways/interaction, dose response, biomarkers, and resistance mechanisms. This article reviews some of the novel information presented at the conference and discusses future avenues of research in this field.

We investigated the importance of the host complement system in the pathogenesis of disease mediated by the intramacrophage pathogen Mycobacterium avium. Mycobacteria opsonized with complement are efficiently ingested by macrophages through various complement receptors. Furthermore, unlike other bacteria, mycobacteria can activate both the alternative and classical complement pathways in the absence of specific antibodies. Therefore, to examine the role of complement in the mycobacterial infection process in vivo, mice deficient in complement component C3 were infected with M. avium. Surprisingly, C3-deficient mice infected intravenously with M. avium displayed no difference in bacterial burden or granulomatous response compared to wild-type control mice. C3-sufficient mice and C3-deficient mice were equally susceptible to infection by M. avium regardless of the genotype at the bcg locus, a locus known to confer susceptibility to infection with intracellular pathogens. In vitro studies using mouse bone marrow-derived macrophages resulted in significant M. avium invasion of macrophages in the absence of C3; however, the kinetics of infection were delayed compared to complement-mediated invasion. The data indicate that complement does not play an essential role in mediating M. avium infections in the mouse and suggest either that other invasion mechanisms can compensate for the absence of complement-mediated entry or that complement is not a major mycobacterial opsonin in vivo.

Cell-based therapies, such as myoblast transfer therapy, are likely to become an integral part of any approach to treat myopathies such as Duchenne muscular dystrophy. Previous studies have shown that an increased level of regeneration in the host muscle enhances incorporation of donor myoblasts. Leukemia inhibitory factor (LIF) increases the number of dystrophic fibers expressing dystrophin after myoblast transplantation and enhances regeneration in injured and diseased muscle. Morphometric analysis was used to investigate whether an increased level of regeneration is induced by LIF after myoblast transplantation. We found that, in muscles treated with LIF, the number of fibers undergoing regeneration was increased. The increased incorporation of donor myoblasts and thus dystrophin expression induced by LIF may be due, at least in part, to an increased level of regeneration of dystrophic muscle., (Copyright 2001 John Wiley & Sons, Inc. Muscle and Nerve 24:695-697, 2001)

Although the muscles of the mdx mouse lack dystrophin, the protein absent in muscles of humans affected with Duchenne muscular dystrophy (DMD), the only mdx muscle to degenerate in a manner similar to those of DMD boys is the diaphragm. We have previously shown that leukemia inhibitory factor (LIF) is a trauma factor that enhances muscle repair in vivo and, when applied exogenously, increases the fiber size of mdx skeletal muscle. Furthermore, we developed a controlled release device for LIF based on a calcium alginate rod (release rate about 0.5% per day). These rods were sutured to the abdominal surface of the hemidiaphragm of mdx mice 3 months old. At age 6 months the mice were killed and the diaphragm muscles fixed and sectioned. The sections showed obvious muscle degeneration at 3 months of age in mdx mouse diaphragms and further degeneration at 6 months in saline-perfused muscle. Hemidiaphragm muscles continuously exposed to LIF over the same period contained more normal myofibers, larger regenerated fibers, and less adipose tissue and other non-contractile tissue. Morphometric analysis of the diaphragm sections was carried out. The LIF-treated animals showed a significant increase in fiber number and size compared to saline rod controls. The amount of nonmuscle (connective tissue and adipose tissue) was significantly reduced and the maximum force-producing capacity of isolated diaphragm muscle strips was higher in LIF-treated mice. The results demonstrate that LIF treatment ameliorates the dystrophic abnormalities in mdx mouse diaphragm., (Copyright 2000 John Wiley & Sons, Inc.)

Growth factors are theoretically promising agents for ALS therapy, but have been disappointing in subcutaneous delivery due to either toxicity or lack of major efficacy. Leukaemia inhibitory factor (LIF), was named after its effect on haemopoietic cells, and belongs to a group of cytokines which includes CNTF, IL-6, CT-1, OM and IL-11. All group members use the gp130 signal transducing subunit for intracellular signalling, but show differences in biological effect. In vitro and in vivo studies on axotomy and nerve crush models demonstrate a powerful effect of LIF in the survival of both motor and sensory neurones, while reducing denervation induced muscle atrophy. Its effects in muscle also include stimulating myoblast proliferation in vitro, and up-regulation after muscle injury. LIF will also stimulate muscle regeneration in vivo when applied exogenously after injury. In published studies of both axotomy induced neuronal death and in the Wobbler mouse models LIF is active at doses of 10 microg/kg delivered systemically, well below the expected maximum tolerated dose suggested by primate safety studies. LIF is expressed in low levels by spinal cord neurones with significant up-regulation when the neurones are damaged by BOAA toxin, an excitatory amino acid associated with a form of ALS. This augments other evidence suggesting LIF is a trauma factor playing a role in the injury response of adult neuronal tissue, and may be more effective than related growth factors. Taken together, the data suggests LIF is a physiologically relevant trophic factor with implications in clinical medicine as a therapy for ALS, and a human recombinant form (AM424), entered human clinical trials during 1998.

A model of spinal trauma was developed where spinal neurones of adult mice were exposed to the excitotoxic glutamate analogue beta-N-oxylamino-L-alanine (L-BOAA). After 24 h, the injured neurones received a single dose of [125I]-LIF at the same site of the spinal cord, and 2 h later, tissues were removed to assess the distribution of leukaemia inhibitory factor (LIF). There was a significant increase in LIF binding to the injured region of the spinal cord over saline controls, and this corresponded with a significant increase in LIF mRNA expression in the same region of the cord. There was a change in the expression of ciliary neurotrophic factor, but the expression of cardiotrophin-1 (CT-1) and the common receptor subunit LIF receptor beta (LIFRbeta) did not change after neurotoxin treatment. The results add to the evidence that LIF plays a significant role in the response of adult neuronal tissue to injury.

Although a number of cytokines have been implicated in tissue regeneration, it is unknown which ones actually function in vivo. Here, we use mice with a targeted mutation in the leukemia inhibitory factor (LIF) gene to examine the role of LIF in muscle regeneration. Using a muscle crush model, we show that muscle regeneration in LIF knockout mice is significantly, reduced compared to control littermates. Further, targeted infusion of LIF in both normal and LIF knockout animals stimulated muscle regeneration, but the stimulation observed was much greater in the mutant animals than in controls. In contrast, interleukin-6 and transforming growth factor-alpha, which also stimulate myoblast proliferation in vitro, had no effect on regeneration. These findings demonstrate directly that LIF is involved in regeneration of injured muscle and points to the use of LIF as a therapeutic agent in the treatment of neuromuscular disease.

Leukaemia inhibitory factor (LIF) has been shown to effectively enhance skeletal muscle regeneration after mechanical injury and it may have potential therapeutic use in the muscular dystrophies as well as peripheral nerve repair after injury. When LIF is applied systemically to an animal, it is rapidly removed with a biological half life of only a few minutes, and at high doses it exhibits toxic effects. Calcium alginate rods have been developed for the purpose of insertion adjacent to skeletal muscles. These rods, when charged with LIF will release the growth factor to the muscle at a rate of less than 1% per day and for a period extending to several months. In addition, tubes of alginate are described which will be suitable for the continuous supply of LIF to repaired peripheral nerve.